JP2002525590A - Support for analyte determination and method for producing the support - Google Patents

Abstract

An electronically-controlled light source matrix (3), preferably a liquid crystal display (LCD) matrix, is faced by a light sensor matrix (9), preferably a charge coupled display (CCD) image sensor. This detects optical behavior of a substance (7) sandwiched between them. Preferred Features: In a related concept, an LCD matrix produces two-dimensional lighting patterns in manufacturing or examination.

Description

DETAILED DESCRIPTION OF THE INVENTION

1. Field of application of the invention 1.1 Background Accurate detection of biologically relevant molecules in defined test materials for basic research and medical diagnostics in biological sciences and some other disciplines. Has prominent significance. Here, the genetic information exists in the form of an enormous variety of nucleic acid sequences of various DNAs. The embodiment of this information, through the production of copies of DNA in RNA,
Mainly protein is synthesized. As proteins they are often involved in biochemical reactions.

[0002] The determination of the order of four bases in a nucleotide chain, referred to as the detection and sequencing of defined nucleic acids, provides useful data for the medicine being studied and used. In medicine, much more in vitro diagnostics (IVD) can be used to develop and provide instruments for determining important patient parameters to physicians. For many diseases, diagnosis is not possible to a sufficiently early point without these devices. At present genetic analysis is an important new method, for example HI
Genetic predisposition for infectious diseases such as V and HBV, certain cancers or another disease;
It has been established for forensics and numerous other applications. In the close association of basic and clinical research, the molecular causes and (pathological) associations of some forms may be traced back to the level of genetic information. But development has only just begun and very thorough efforts are needed right now to switch to treatment strategies. Overall, genomics and the subsequent analysis of nucleic acids make a great contribution to understanding the molecular basis of life as well as to very complex pathological and pathological processes.

[0003] Other developments in the drug supply are burdened by the exponential increase in costs following the correspondingly expensive methods. This should not only drive the realization of diagnostic and therapeutic feasibility, but also promote its integration into affordable medical systems.

[0004] The use of appropriate technology in research can only be done extensively, and only in universities, if the costs involved are reduced.

1.2 Demand [0005] Advances in genomics and protein sciences and the determination of overall genetic disposition are still at the beginning of development and are also at the stage of realizing the diagnostic potential of genetic or genetic engineering analysis. The methods established up to now are mainly costly to work and relatively inefficient, as reflected in, for example, the cost and ability to acquire information. A significant innovation is the development of so-called oligonucleotide arrays, in which a very large number of relatively short oligonucleotides of a defined sequence are coupled onto a solid matrix (mainly silicon), And thereby provide for parallel hybridization of complementary sequences in the test material. However, expensive manufacturing and high prices cannot be marketed as mass products at this time.

1.3 Fields of Use Oncology systems are relatively inexpensive for in vitro and clinical diagnosis, for example for infectious diseases (HIV, HBV, etc.) and their subtypes, oncology (early tumor detection,
For tumor classification, eg, type and condition) and for determination of genetic predisposition,
Should allow for repeated use.

[0007] For basic biological studies, in particular genetics, it is desirable to include a very large number of measuring points in the system under investigation, for example in all test genes. From now on, a great deal of knowledge will be acquired in biological basic research (development biology, stem cell culture, tissue engineering, transplantation medicine, regeneration)
And the study also leads to important breakthroughs and corresponding uses in biomedicine.

As indicated for the use of DNA chips (Science 280: 10
77-1082), which may be distinguished from the corresponding biochemical limitations of hybridization between point mutations in base order. Further, with respect to the systems described herein, it allows for a wide range of screenings that can be used for legal purposes, such as criminal evidence or proof of kinship.

[0009] Also according to the invention, a fast and cost-effective analysis of foodstuffs is possible, for example, based on the presence of defined genes of pathogenic bacteria or genetically altered organisms.

[0010] Similarly, screening for medical products is very significant. For example, the production of blood products is still associated with high expenses for safety precautions to purity. Time- and cost-effective screening of such samples is possible according to the invention, for example to prevent contamination by infectious substances (HIV, HBV, HCV, etc.).

[0011] 2. BACKGROUND OF THE INVENTION Biochips comprise biological and industrial components, such as biomaterials (e.g., DNA-oligonucleotides) and silicon matrices that can be used as specific interaction partners immobilized on the surface of a support. It is a miniaturized hybrid functional element having the same. Primarily, these functional elements are arranged vertically and horizontally, which is called a biochip array. If thousands of biochemical functional elements can be arranged on a biochip, they must be manufactured by microtechnological methods.

In particular, in the United States, the development of miniaturized biochips has been advanced by many methods. The most important companies operating in this environment are listed below: Affymetrix, Beckman Instruments, Bluechip Biosystems, Caliper Technologies, Klagen, Genometrix, Gentrail Systems, Hysek, Insight Pharmaceuticals Callus, Molecular Tools, Nanogen, Pharmacia, Syntenai, Third Wave Technologies, Weissis.

[0013] Biochips known in the art can be classified by the following criteria: * Principles of detection-Chromatographic methods-Interaction of analytes with solid phases, mainly immobilized interaction partners (eg DNA Hybridization of nucleic acids to oligonucleotides) * Detection methods (optical, electrical) * Label-based detection methods (eg, adsorption, fluorescence or luminescence) or label-free detection methods (luminescence for detection of reactions) * Assignment of analyte to its support (solid phase) (array with more than one immobilized interaction partner per support or only one immobilized interaction per support) Single (with partner) * Manufacturing method (eg, oligonucleotides are photoactively synthesized directly on biochips and fully synthesized oligonucleotides Covers beads or tubes interspersed with otide) * Type of support (glass chip, plastic chip, microtiter plate, tube or bead) * Display for detection (serial, parallel) * Optical Target detection (either serially with a scanner or in parallel with a CCD camera).

Among the companies mentioned above, only Affymetrix is the DNA on one flat surface
It uses the principle of photolithography for "dense" production of arrays. For this reason, the company is leading the way in parallelizing the detection of oligo sequences by a large margin.

GenChip (Affymetrix Inc, Santa Clara, Calif.) Its production is performed at high density on a flat chip by in situ synthesis of DNA oligonucleotides (July '98: 64000 different species per cm ² ). Oligo). Based on photolithography used and optimized in the semiconductor industry, the oligos bind photoactively to the chip surface as well as to the already existing oligos. Manufacturing time is a few hours based on a number of process steps. Detection is performed in a fluorescence scanner by serial optical detection of a flat chip. The hybridization time of the sample on the chip is about 1.5 hours. The first products (tumor marker p53 exons 2-11, sequencing chip for breast cancer gene BRCA1 exon 11, HIV gene chip) are already commercially available. Prices are currently in the hundreds of dollars range for gene chips, but detection equipment is also required.

[0016] Further relevant prior art is WO 91/18276, EP-A-0671626.
And EP-A-0430248.

[0017] 3. Object of the invention and problems solved by it An object of the invention is a method according to claim 1 for producing a support for the determination of an analyte, the method comprising the steps of: Providing a support having passages; (b) conducting a liquid having a component for the synthesis of a polymer receptor through one or more passages of the support; and (c) connecting one or more receptor components to the support. Immobilizing position-specifically and / or time-specifically at each of the predefined locations or regions of the passageway (d) until the desired receptor is synthesized at each of the predefined locations or regions (B) and a step of repeating the step (c).

[0018] Advantageous embodiments of the method are the object of any one of claims 2 to 13. The support is a biologically or chemically functional material or receptor (probe) or a solid phase capable of being equipped with or equipped with these components. In the above embodiment of the invention, the support has a cavity, for example a surface having at least one passage and particularly preferably a large number of passages. These channels are preferably microchannels having a cross section of, for example, 10 to 1000 μm. These passages may be capillary passages (depending on surface properties), but may also be passages without capillary action (e.g. based on coating with Teflon). The support is advantageously at least partially optically transparent in the region of the location or area in which the receptor is provided. The areas of the support on which the receptors are equipped are advantageously chemically and physically identical to one another. That is, they have substantially the same surface properties.

A further object of the present invention is the method according to claim 14 for integrated synthesis and analyte determination on a support, said method comprising the steps of: (a) providing a support. (B) directing a liquid having components for the synthesis of the containing receptor or polymer receptor onto a support; (c) also pre-defined positions of the receptor or receptor components on the support. Or position-specifically and / or time-specific immobilization at each of the regions, (d) optionally until the desired receptor is synthesized at each of the predefined positions or regions on the support. Repeating steps (b) and (c), (e) contacting the sample containing the analyte to be measured with the support, (f) transferring the analyte onto a support on which the analyte is immobilized. Comprising the step of measuring by the binding to data.

An advantageous embodiment of the method is the object of any one of claims 15 to 25. A flat support may also be used in this embodiment.

The object of claim 26 is to provide at least one passage and advantageously a plurality of passages,
It is a support for measuring analytes, in particular having a plurality of capillary passages, in which a number of different receptors are immobilized. Advantageously, the support is optically transparent at least in the region of the area equipped with the receptor.

A further object of the present invention is a reagent kit according to claim 28, comprising a support as described above and a component for the synthesis of a polymer receptor on a support. In addition, this reagent kit even contains a reaction solution for the synthesis of the receptor on the support.

Also an object of the present invention is an apparatus for integrated synthesis and analyte determination on a support according to claim 29, the apparatus comprising a programmable light source matrix, a detection matrix and a light source matrix. And a support disposed between the substrate and the detection matrix, and means for supplying liquid into the support and discharging liquid from the support.
The programmable light source matrix or illumination matrix may be a reflective matrix, a light valve matrix, such as an LCD matrix or a self-luminous illumination matrix. Advantageous embodiments of the device are the objects of claims 30 and 31.

Finally, it is an object of the present invention to use the claimed method, support, reagent kit and claimed device for the determination of an analyte in a sample. An advantageous use is the object of any one of claims 33 to 38.

Embodiments of the present invention shall mean methods and systems for periodic integrated synthesis and analysis, which shall mean ISA systems. The direct integration of synthesis and analysis advantageous according to the invention enables a high throughput measurement of analytes which is clearly improved over the prior art in a cyclically proceeding manner. In that case, the substance to be analyzed may be present, for example, as fragments or fragments of a polymer chain.

According to an advantageous embodiment of the invention, there is provided a direct logical connection between the analysis of the first support and the synthesis of the subsequently produced support, wherein the previous Information acquisition from a cycle can be diverted to a subsequent cycle. As described above, the learning of the analysis system is gradually developed.

The order of the cyclically proceeding synthesis, sequence comparison, analysis of the results of the comparison and the synthesis of the receptor on the support, which is carried out again, is arbitrarily often (depending on the desired arbitrarily chosen interruption criteria). Until it reaches).

By means of the learning process of the cycle before the return and its connection, the method and the device according to the invention make it possible for the study of very large and complex analytical molecular chains, for example for the sequencing of individual genomes, for example the human genome of an individual. Appropriate. The time expenditure is then at least 100-fold, rather 1000-fold, and potentially 1-fold over the prior art.
0000-fold improvement.

The method comprises the steps of “novel sequencing” of unknown nucleic acid sequences (DNA, cDNA, RNA).
For these, these spatial distributions, including their respective mappings, can be used. In this manner, individual and various individual gene profiles can also be generated by sequencing parts of the genome or the entire genome.

Furthermore, the method can be used for “re-sequencing” of nucleic acid sequences. That is, it can be used for comparison of an already known sequence (expressed in the form of a receptor probe) with an unknown sequence in the sample to be investigated. The known sequence is chosen for this purpose and depending on the question.

[0031] Said resequencing allows the user to generate individual polymer receptors in situ on a support according to the invention from a neutral support and to immediately carry out the analysis of the sample to be investigated. Make it possible. This possibility results in maximum receptor variant diversity with minimum requirements.

The combination of novel sequencing and resequencing allows a diagnostic test or drug to be quickly adapted to the needs of the individual.

As a further important field of application, extremely flexible phenotypes can be analyzed. To that end, the corresponding receptor or polymer probe is selected on the basis of generally known sequences. Use of the method for determining gene expression can also be performed for high-throughput screening.

In addition, different batches of various naturally occurring and artificial receptor probes can be envisioned for screening methods, and for constructing and analyzing material libraries. This can be done, for example, in connection with the search and characterization of pharmacologically active substances.

The method according to the invention and the method according to the invention and the device according to the invention for periodic integrated synthesis and determination of analytes are broadly divided and in principle include gas chromatography, thin-layer chromatography, Relevant for all uses of analysis, such as gel electrophoresis, capillary electrophoresis, mass spectrometry and the like. A similar one is
In principle, it is regarded as a highly parallel use of all solid-phase analyses.

The need to mount off-the-shelf complex polymer receptors is completely eliminated. Furthermore, there are no restrictions on the total number and selection of receptors. The required total number of receptors can be distributed over a plurality of reaction supports or on one reaction support over a plurality of cycles. This is because the individual receptors are not subject to the location criteria for the logical evaluation of the comparison result.

It is an object of the present invention to form highly parallel, highly flat and dense arrangements (arrays) in a solid support matrix (chip), preferably with individual bases (arrays).
G, A, C and T) or a new "support" as a basis for the use of light-controlled synthesis of all oligonucleotides (sequences).

The novel biochip, an “optical fluid microprocessor”, has a structure that is at least partially transparent and consists of microchannels, preferably capillaries, in a preferably flat object. The liquid use substance is introduced into the support through the channel during receptor synthesis or receptor immobilization and is locally activated and bound to the channel wall. This fulfills the technical prerequisites for rapid, effective and thereby inexpensive production, and allows the support to be used in a wide range. Density and parallelism are on the same order as competing technologies, with hundreds of thousands of defined oligonucleotides on the support.
An advantage of the new technology is the advantageous physicochemical properties of the flow process and the wetting process in multiple passages compared to a uniform surface.

The production of the chip advantageously comprises the production of a support made of a suitable light-transmissive material with microchannels and the process of biochemical coating the individual microchannel walls.
Subsequently, polymer receptors, eg, oligonucleotides, can be synthesized in multiple passages. The individual receptor constituents, oligomeric synthons (eg dinucleotides, trinucleotides, tetranucleotides or pentanucleotides) or the complete base sequences (oligos) are then activated in the individual passages in the support by means of a suitable light source. Are site-specifically attached. This results in regions (specific binding or hybridization sites) equipped with multiple receptors in each passage, wherein the individual regions based on the respective receptor sequence set are specific analytes, such as DNA fragments. Useful for binding and subsequent detection. The areas are separated from one another on one side of the flat support by a plurality of channel walls, and a corresponding space remains between the two adjacent areas along the individual channels in a photoactive coupling. A very parallel, highly integrated array of specific receptors results. Based on the possibility of multiple use of oligo sequences and parallel pathways (see Chapter 5 for details), the production time is reduced by a factor of 4 when using a single base and by 1 when using a dinucleotide. / 8, and 1/16 when using trinucleotides, can be shortened by the corresponding multiple use of oligos (substances used) and a plurality of wet passages. This also allows for flexible adaptation to customer requirements ("custom-made" biochips). These systematic accelerations are not possible in flat systems (flat chips).

For analysis, the investigational material (eg, DNA, RNA in solution) is introduced through multiple passages, and the opportunity for binding to the receptor is obtained, for example, by hybridization to the complementary strand, if present. . For the detection and evaluation of the respective analyte binding, for example DNA hybridization, a high-resolution parallel CCD chip is advantageously used. The binding of the analyte to the immobilized receptor is via a suitable signal-donating group known from the prior art, for example a luminescent group. However, new detection methods can also be used. If the size of the passages is chosen such that each measuring point covers a sufficient number of detectors, for example the pixel elements of the CCD chip, an optically imaging lens system cannot be used in the detection. Number (16Mio pixel current per 1 cm ^2; Level 1 cm ² per 80Mio pixels studies) very parallel with the pixels (optical sensor) (not optically) C
Multiple light signals can be detected in parallel by using a CD matrix chip directly (see BioScanner from Genometrix). At the same time, attempts have been made to use large and inexpensive high-tech products instead of expensive optical arrangements in the detection device.

Thus, according to the present invention, a substantial requirement in DNA analysis, ie, a large number of DNs
Simultaneous measurement of A-arrays (achieved by highly integrated miniaturized supports and high-resolution optical detection), providing inexpensive testing (multiple use in manufacturing, inexpensive "disposable", eg injection molding Support, rapid synthesis in production)
, Rapid performance in small volume based analysis and reduction of materials used due to advantageous wetting processes, flow geometry of supports, etc., rapid evaluation (flat configuration [DNA
Achieved by parallel optical evaluation in the chip], inexpensive analysis systems (achieved by expensive microsystem technology and elimination of optical components)
As well as quality assurance in the analysis as well as in the production (achieved by defined flow processes in the support) are covered.

The use of the photoactivity of chemical reactions in the area of support synthesis results in a combination of the technical basis of a photofluidic microprocessor and a programmable light source matrix, among others. This is because the manufacturing costs for the individual supports can be improved at the same time by a factor of about 10 to 100 times. This makes it possible for the first time to obtain inexpensive, highly parallel, highly integrated and easily miniaturized and automatable DNs
A chip-technology is provided.

Despite complex data evaluation, only a minimum of one of the various hardware components is required. This is because the supports that need to be replaced on a cycle-by-cycle basis or only during wear are all the same before the start of receptor synthesis. All attributes are first turned into information after specific receptor synthesis and synthesis / analysis cycles,
The nature, that is, the characteristics of biological / chemical substances, exists only in the form of electrical data, on the contrary, as it comes from the information gained by the analysis.

4. Basic route of the solution The principle solution in this system is based on the stepwise biochemical synthesis of receptors on the surface of a number of passage walls on a support. These passages are arranged on a support, for example a small flat chip. The synthesis is carried out by photoactivated regiospecific binding with an oligonucleotide (base sequence) of the appropriate base or of a plurality of bases. The wetting of the passageway specifically "labeled" with the DNA analyte to be investigated and the subsequent detection of the binding reaction by the signal-donating group closes one process cycle.

4.1 Microstructure as Support Matrix Support synthesis involves the preparation of a support consisting of a suitable light transmissive material and the biochemical generation of receptors on the individual passage walls. The specific synthesis of the receptor can be carried out directly during the production of the support or on first use.

Various materials can be used for the support (eg, glass, silicon, ceramic, metal or plastic). It is important that the passage walls are as well as transparent to the light waves (and possibly the excitation and reaction signals) in the subsequent detection (analysis) as well as the excitation waves in the photoactive synthesis. Depending on the use of the material, the passage walls must be coated with a reactive material, so that the receptor or the receptor component can bind to the surface.

The geometry of the support corresponds for example to a “cash card”, wherein the size of the surface covered by the passage is defined by the CCD chip used for the detection. Various manufacturing methods can be used for the plurality of passages in the support. that time,
The influence of the cross-sectional geometry of the multiple passages is taken into account, which has a significant effect on the corresponding flow engineering forces and has the cleaning capability of the multiple passages.
As a manufacturing method, for example, laser, milling, etching or injection molding can be used.

The following aspects can be considered in the arrangement of a plurality of passages in a plane: if a large number of parallel passages are used, the synthesis time can be reduced, but the wetting or filling of the individual passages is correspondingly complicated. It is. With extremely only one single long passage, the synthesis is correspondingly slow. This is because multiple use of multiple passages cannot be used for bases or all oligos, and all processes can proceed one after another serially only. The advantage of only one passage lies in the analysis in which the sample is pre-flowed in all passages at each measurement point.

4.2 Synthetic Cycle in Support The location (reaction zone) on the support intended for coating with the receptor is one or more of the liquids via conduits, valves and fittings from the vessel through multiple passages. Will be filled. Using an illumination / detection device which means a light source matrix or an illumination matrix as known from German patent application 198 35 254.0, preferably as described in German patent application 19907080.6, A selected position or area of the support can be illuminated, thus controlling the individual response of the receptor, the associated support then being an optical fluid microprocessor. Instead of irradiation, individual fluid activation of selected reaction zones is also possible. After the completion of the reaction, the reaction zone is flushed and refilled, after which the reaction cycle is resumed. The process of receptor synthesis can be performed and controlled using suitable detection equipment.

As soon as the synthesis of the receptor has ended, the reaction zone is cleaned and an analyte measurement process is provided.

4.3 Nucleic Acid Analysis with Oligo Chips-Basic Principle As already shown in a number of arrangements (eg Molecular Medicine Today, 9/97, pp. 384-389; Trend in Biotechnology, 1
1/97, pp. 465-468), mainly hybridizing complementary sequences, so-called oligonucleotides or oligos, to nucleic acid strands can be used for sequence analysis. In addition, a high density arrangement of synthetic oligonucleotides occurs on the solid matrix and allows for a variety of parallel hybridization tests. Tip method (
(August 1998) is photolithography at the pre-synthesis stage and its activation. In reliance on technology borrowed from microelectronic manufacturing, the parallel arrangement is called a chip.

Large analytical capacity is achieved by a large increase in the number of reaction zones (“measurement points”), ie, defined oligos at defined positions.

The sample to be investigated usually contains DNA or RNA. It is optionally isolated and amplified by an amplification step (eg PCR) and receives a label, for example a dye label, a fluorescent label or a luminescent label.

DNA molecules can also be sequenced by regions equipped with a sufficient number of receptors (reaction regions) (sequencing-by-hybridization SBH, Bi
oTec3 / 98, pp. 52-58), a further use is point mutation-polymorphism (
Ie, differences between individuals of several bases in a given DNA fragment), making it possible in particular to identify such polymorphisms in several hundred subjects in parallel (Science 280, 5/98, 1077). -1082 pages).

It is also possible to investigate the whole genome and the state of gene expression in all cells (for example, Proc, Nat. Acad. Sci. USA 95, 3/98, S. 3752-3757).

The invention described herein thus allows the use of a number of established methods for the investigation of nucleic acids and genetic material. Moreover, at the same time, a great increase in such uses and consequently a great scientific research is linked. This is because optofluidic microprocessors are expected to provide more flexible technology and significantly less cost than existing methods.

4.4 Photoactive Synthesis of Oligonucleotides and Peptides on the Support During the construction of the receptor on the support, the receptor components, for example the individual bases, are activated by photoactivation by means of a suitable light source in the individual regions. (G, A, C, T) or an oligonucleotide sequence (preferably about 2 to 4 bases in length) is added regiospecifically.
The multiple paths are sequentially filled with synthetic components, eg, G, A, C, and T, and illuminated along the multiple paths with position-specific, high-resolution light of a defined wavelength and intensity.
During the coating cycle, the channels are flushed accordingly to remove unbound receptor components.

At this time, a large number of reaction regions (specific binding sites or hybridization sites) are generated in each passage, and each reaction region is based on its individual receptor sequence, and has a specific analyte, such as a DNA fragment. Useful for binding and subsequent detection. The reaction zones are separated from one another by a plurality of channel walls in a first dimension of the flat support and in a second dimension upon light activation between two adjacent reaction zones along individual channels. A reasonable space remains.

Photolithography can also be used for photoactive coupling of receptor components. However, other methods may be used.

It is particularly advantageous to carry out the irradiation process using a programmable light source matrix, for example a self-luminous light source matrix, a light valve matrix or a reflection matrix,
The matrix points or light source elements are intended to be controllable, in particular with respect to the intensity of the light and possibly the color of the light. Using such a matrix, the respective required two-dimensional illumination pattern can be easily created, in particular with the aid of a computer. The advantageous photoactivation of the oligos during the production of the support is effected directly by the irradiation matrix. For example, a wavelength of 365 nm (
The upper UV region near the visible light) is controllable by all variants of the programmable light source matrix.

[0061] Accordingly, the receptor can be composed of amino acid and / or peptide components.

4.5 CCD-Chip Detection of Specific Detection Reactions As described above, binding of a DNA analyte is directly or indirectly detectable signal,
For example, it should be guided by a light signal. This includes, for example, adsorption, excitation light (fluorescence)
Alternatively, it can be implemented by photon emission (emission). Advantageously for signal detection,
Preference is given to using a CCD chip which is arranged directly below the support. The excitation light source is
It is preferably arranged on a support and measured correspondingly by transmitted light.

Each light signal can be recognized on a CCD chip, and is further distinguished by intensity and, if necessary, wavelength (color). The received spectrum can be evaluated qualitatively or quantitatively. Further, the distinction between wavelength and intensity also allows distinction between signal sources.

As the type of excitation light for the detection method, monochromaticity (for example, laser light for fluorescence excitation) or heterogeneity (for example, white light for adsorption measurement) is selected depending on the arrangement.

[0065] 5. Improvements and Benefits over Existing Systems The novel support overcomes the following disadvantages of masking-based photolithographic methods or in-situ spotten.

* The principle of flat wetting of the entire chip surface with liquid does not allow any kind of multi-usability in manufacturing. Therefore the number of production cycles for 20 base long oligonucleotide is increased by the use of dinucleotide ⁽⁴ possibilities 2 = 16) 4 × 20 = 80 for the hybridization step to the 16 × 10 = 160, which is Means doubling. Of course, it also corresponds to an intervening cleaning cycle.

* Synthesis of photoactivatable bases on a flat chip surface, as well as washing steps required during chip manufacture are location- and operation-intensive dipping processes (dipping the chip in liquid) or liquid This can only be achieved by means of intensive surface flushing processes (eg the principle of centrifugation from semiconductor technology), which hinders very large miniaturizations and automation which are favorable from the development of the equipment.

In subsequent DNA sequence detection, it is expensive to distribute the sample evenly over the chip surface (easy and at the same time no reliable mixing method is possible) and correspondingly large Of sample solution is required. It is impossible to look for rare events in a sample. This is because sufficient contact between all sample components and all specific measurement points cannot be guaranteed.

5.1 Shortening of Production Time by Multiple Uses during Synthesis A substantial development of the new supports is the use of receptor components and multiple passages in the individual synthesis of receptors equipped with receptors. The corresponding multiplexing can drastically reduce the production time.

DNA-sites for the site-specific generation of a large number of different receptor sequences, eg, nucleotide sequences of defined length (eg, 20 bases), by positionally high resolution photoactivation on flat surfaces. 4 in each region of the chip array (calculation example: 20 bases in each base sequence)
One synthesis cycle (caused by four different bases) is required. For a hierarchy of 20 bases, therefore, 4 × 20 = 80 cycles. Dinucleotides on the same surface (
When (2 bases) is used, two hierarchies occur at one time, however, these two hierarchies require a synthesis cycle of 4 ² = 16. Thus for 80 levels 80
Instead of a period, a synthesis period of 10 × 16 = 160 is required, which means a doubling of the production time. In the use of trinucleotides (3 bases), these effects increase cycle number by more than 5-fold. Therefore, the light-activated D on a single flat surface
The fastest possibility for making NA chips involves the use of a single base. There is no possibility of reducing the number of synthesis cycles.

Alternatively, in the synthesis of the optofluidic support, the substances used, ie bases or dinucleotides (combination of 4 ² = 16) or trinucleotides (4 ³ =
64 combinations) can be distributed to different passages. That is, at least in the lower “near the support” hierarchy, only one base or one of the possible base sequences is incorporated per path. Depending on the definition of the total base sequence occurring in the multiple passages of the support, in the upper hierarchy the principle should be partially negated, i.e. for base-plane, dinucleotide-plane or trinucleotide plane. Requires more than one base or oligo to flow through one of the multiple passages. This also increases the number of synthesis cycles somewhat again.
However, a very large reduction in the overall production time is due to the fact that the use of substances in receptor synthesis is theoretically 1/4 of the period for a single base, 1/8 of the period for a dinucleotide, and When using trinucleotides, it remains at 1/16 of the cycle (such as in longer oligos). The number of cycles required for a specific support can be determined if the number of reaction zones on or in the support, the number of parallel pathways and the length of the oligo synthesized on the support are specified. It is individual for each support and can only be determined as a statistical average. Optimization of the synthesis time of the support can be accomplished by using developed software tools (eg, CAMS Computer Aided Multiplexing Synthes).
i). This software tool is incorporated into the controller of the developed analysis system or a connected computer.

5.2 Reduction of Materials Used and Quality Assurance The use of multiple passages reduces the required liquid volume and at the same time the quality is reduced to the use of a single surface both in support synthesis and subsequent sample detection. It is extremely enhanced compared to. The simultaneous wetting of several passages is therefore very easy in terms of flow technology, the consumption of liquid is low and therefore miniaturization and automation are very easy. In particular, this also applies with sufficient quality to the required cleaning process of multiple passages.

The required liquid is reduced to almost 50% by a plurality of passage walls covering the space between the two reaction areas in the support array. This applies to "coating samples" for analysis as well as to coating of supports during manufacture, synthesis of receptors. A further reduction of the liquid volume is achieved by a good wetting of the channel walls by the liquid flowing through and by a particularly effective cleaning process. This cleaning process can be significantly improved by, for example, "cleaning" bubbles in a plurality of passages. On the surface, a statistically sufficient good distribution of the sample on the surface can only be achieved with a very large amount of sample.

A further advantage of the multiple passages is a shorter cycle time, which is provided by a smaller liquid volume and a subsequent rapid chemical reaction and completion. This results in both short synthesis and hybridization times.

Furthermore, a clear reduction in defects is achieved in the detection as well as in the production, which further increases the number of measurements that can be evaluated with each use of the material and time, and provides a basis for quality assurance. To form This basis makes up a flow process that is precisely definable and reproducible.

The easy miniaturization and automation of the outlet in the new support forms the basis for easy miniaturization and automation of the whole new analytical system built on the support.

5.3 Three-Dimensional Reaction Surfaces By appropriate design of the cross-sectional geometry of the individual passages, the available reaction surfaces can be expanded. The size of the surface is as important for the addition of oligos during production as the intensity of the light signal in the addition of pre-flowed DNA fragments from the sample and the resulting hybridization performed.

The rectangular passage, which therefore requires the same height as the width, has a reaction surface that is four times as large at the same base area, ie at the same required area in two planes of the flat support, due to the wetting of the walls and the deck. Having. Even if the passages are designed to be rounded internally due to flow engineering requirements (eg good cleanability by air bubbles in the passages), approximately three times the reaction surface compared to a flat surface can be increased. Hold. The use of materials (production and analysis) can be further reduced by using this three-dimensional flow geometry.

Further effects can likewise be influenced by the cross-sectional geometry of the channels: refraction of light during the transition of the internal space of the channels in the surrounding material of the support. Therefore, each curvature has a focusing action or a scattering action in the light propagation direction. The light path can be optimized in the support by a corresponding choice of the upper and lower flow path geometry.

5.4 Parallel CCD Chip Detection Measuring the light signal of all reaction areas “at once” requires a high resolution CCD
A permanently increasing voltage of the camera is used. This allows the detection of all light signals for reaction detection or hybridization detection in a single measurement process. For this purpose, a modern color CCD on a 40 × 40 mm surface
The chip provides about 3000 × 3000 pixels with a pixel size of about 10 × 10 μm. The level of research has about 4000 × 6000 pixels for a corresponding CCD chip. Signal detection is performed simultaneously for all pixels in an instant. At the same time cause significant growth potential with regard applications described in the CCD chip technology, parallel detection of 10 ⁶ individual reaction regions in the support it can be realized technically. This avoids the time-consuming scanning process of commercial systems,
And the net measurement time is reduced to a minimum, which is completely insignificant when compared to other process steps.

The processing of the resulting data volume is possible without problems due to price destruction at the same time with the development of the performance of modern computer systems.

5.5 Direct detection without optics Direct detection of the light signal by a CCD chip without optics has the advantage of a substantially lower energy content, which can be detected without errors. Need light for Such an arrangement (tested in another context) uses only 10% of the excitation light of a comparable arrangement with optics. In other words, the optical system consumes 90% of the light energy. Due to the low light intensity, undesired scattering effects in the support (around the passage) are significantly reduced, as is the cooling required for the light source used. Furthermore, the elimination of the optics means a large space saving and a reduction in the manufacturing costs for the detection device.

[0083] Expensive, motion or detection devices for the support required in the scanner are likewise completely eliminated. The defined dimensions of the CCD chip (several cm ² ) allow the use of a large number of parallel paths with a reasonable path size (range 10-100 μm).

5.6 Disposable Supports These supports can also be finished as a single-use disposable tip. In principle, glass chips, silicon chips, metal chips, ceramic chips or plastic chips (inexpensive injection molding methods) and other implementations are also possible.

Another technology biochip is similarly interpreted as a disposable for small measurements. However, here, based on the expensive manufacture of chips,
Very high costs have been reported for disposable tips after only one or a few measurements.

5.7 Flexibility of Use Fast and inexpensive manufacture allows for a variety of individual uses. With regard to this use, the synthesis of oligonucleotide arrays is intended in a centralized manner, taking into account, for example, sequence banks and gene data banks on the Internet.

Hybridization is achieved with (slow) flow-through by the use of a single, multiply-wound or spiral-shaped passage, allowing the detection of rare events (eg rare measurement genes). At the same time, the principle of chromatography was introduced into DNA array technology.

[0088] Further reductions in production time can be achieved by the use of dinucleotides, trinucleotides or longer oligonucleotides as synthetic components. In particular, for simpler arrays, the synthesizer can be used directly on the customer side, and furthermore, the composition of the array can ultimately be individualized.

The great adaptability of the technology is also important with regard to recognition, in that each individual's genes differ very significantly, and as a result fit into the general gene catalog for all species. The support has the ability to match the base data, which is freely available on the Internet or exclusively sourced for taxonomy only, for example in the first measurement cycle, by individual patient differences and from the results the actual test to the subject This opens up the ability to form a corresponding second DNA array.

The solution according to the invention can also be used in multiple channels for the synthesis of peptide sequences. This provides a very complex and simultaneously inexpensive peptide array for multiple uses.

[0091] 6. Perspectives According to Some Aspects of the Present Invention 6.1 Support Variations In a configuration similar to support shaping, a number of variations exist. In the arrangement of the passages in the support on the surface of the detection device, a single passage can be envisaged as well as an arrangement of multiple parallel passages. A 500 mm having a length of 37 mm and 750 reaction zones each on a single 25 × 37 mm surface without problems in terms of manufacturing technology.
Passages (prior art: 500 parallel capillaries with a diameter of 900 nm). The same number of reaction zones (500 × 750 = 375000) can be accommodated in a single coiled passage having a length of about 20 m.

The advantage of only one passage is that the sample is present at every measurement point in the array,
Therefore, it is particularly suitable for searching for rare components. The large number of parallel passages has the advantage that the production time in the synthesis of the support can be minimized by the multiple use of the materials used and the passages and by all the flow processes. The passage arrangement is therefore advantageous for support synthesis as well as for all analyzes with a sufficient number of copies of the analyte in each sample.

In order to use the advantages of both in one support, the material to be used may be divided into multiple passages during the synthesis of the support, even though the passage for sample administration consists of only a single long micropassage. Introduced by fittings in parallel to the entrance to the. This effect can also be implemented by introducing a valve into the support or instrument component. Accordingly, Biacore has realized a valve that is fluidly controllable on a two-part injection-molded chip by means of a membrane which presses from below the plurality of passages to above the chip, thus closing the plurality of passages.

Numerous structures and micro-passage paths are possible as arrangements for the passage on the detection surface. Due to the high parallelism of the flow process, for example, parallel or "coiled" structures are easy. In this case, the distribution of the plurality of passages is performed based on the dual principle. This means that two new paths result from each path and that all paths are of the same length. In this way, 2 ¹⁰ = 204 has already been obtained by dividing into ^ten.
Reach eight passages. The coiled arrangement has the advantage that the arrangement has a slightly turbulent flow process and can be better cleaned. These major disadvantages are:
A supply or discharge that must be carried out up and down in dimensions, which is rather inconvenient in manufacturing technology and optically.

As the material of the support, for example, glass, silicon, ceramic, metal and / or
Or plastic is possible. The construction is done in two layers, with or without contact with each other, for example by gluing or bonding. In this case, the structure of the passage may be provided on one side only, or on both sides or half. For this purpose, laser or precision milling can be used as a production method, among others.
Injection molding is particularly inexpensive and can be manufactured with sufficient quality. Another method is LI
GA technology or thermoforming.

6.2 Support Synthesis There are in principle two possibilities for synthesizing individual capture receptors, such as oligos, on reaction areas in a support array. The customer obtains the completed support from the manufacturer by selecting the immobilized base sequence, or synthesizes a self-selective sequence on the unlabeled support in a synthesizer. Information relating to the corresponding sequence can be obtained, for example, from a databank on the Internet, and this information is free here,
Or provided specially by the manufacturer of the support.

6.2.1 Synthesizer The synthesizer consists of a suitable light source, which reacts the reaction area in the support array with a receptor, for example a base or a base sequence during the synthesis of a base or base sequence. Illuminate with high precision and precise resolution on a wall in a position-specific manner. As already mentioned in 4.4, irradiation can also be performed by a programmable light source matrix. When using a photolithographic apparatus, it can be used as in semiconductor chip production for photoactive etching of Si wafers.

6.2.2 Synthesis of the Completed Support by the Manufacturing Machine The synthesis by the manufacturing machine is carried out during the manufacture of the finished support. This requires a correspondingly efficient synthesizer, which uses as long as possible oligos (3 or more bases in length), which are introduced in parallel in a plurality of passages ( Injection), thus reducing the production time per support (multi-use).
In this case, a specific inlet can be provided in the support, the intention of which is to provide as many parallel and thereby short passages as possible, without providing a passage structure for the analytical process. You.

6.2.3 Materials Used in the Support For uses where the construction of the individual arrays is important, not depending on the rapid synthesis of the support, the materials used (G, A, C, T and buffers) Preparations are possible directly on the support in a corresponding storage. The extra material used must be accommodated in a room in the corresponding support. The capacity of such a room can be designed to be many times larger than the total passage capacity without any problem by three-dimensional expansion up and down. The use of a variant of the support can be envisaged especially for a laboratory, but also for small etching implementations.

In this case, the principle of capillary force can be used for liquid transport in the support directly in a possible variant. Without any mechanism, the filling of the capillary with the substance used and the sample can be carried out by easy adjustment of the valves in the support. The "waste room" has an absorption effect facilitated by the embedding of a suitable fleece material. To achieve the minimization of the required liquid volume, a constant length of capillary should be taken into account during the discharge of the unrecovered stream (not circulated and therefore the material used is not reused). This is also important as a pump for the function of capillary forces.

Since another variation is the vertical adjustment of a flat support, gravity can also be used for liquid transport in the support. If these forces are not sufficient to achieve all the required liquid transport in the support, another suitable pumping mechanism can be provided. This possibility is due to electrodes (incorporated in the support) or external to the support (classical pump)
Electrophoretic movement of the liquid due to a reduction in the volume of the support chamber due to a corresponding force action from the liquid.

6.2.4 Use Materials in the Synthesizer The preparation of the use materials for the synthesis of the support in a storage vessel offers in principle the advantages of the complete sequence and the multiple use of parallel pathways. Therefore, these (ultra) high throughput screening and variants for the support maker are worthy of recommendation. The multiple use takes place at the point of contact with the support. Here, a particular base sequence wets another passage for each synthesis cycle. A technically expensive, but sometimes reliable, method is the multiple use in a device with a corresponding valve system. Here, consideration should be given to possible substitutions caused by the use of various base sequences.

Another point that must be taken into account is to contain and remove excess material at the exit of the individual passages. Here, both circulation (reuse of the separated materials) and removal of the separated used substances can be envisaged.

6.3 Analyte Determination Analysis of nucleic acid sequences is performed by hybridization of nucleic acids in the sample material, as in another oligonucleotide array, to strands complementary under the immobilized oligonucleotides. .

As another possible use of the support, the peptide sequence can be coupled in multiple passages, also by the principle of in situ synthesis. The range of potential analytes can be significantly expanded because such peptides are capable of binding reactions with peptides, proteins and other substances that are diverse and partially highly specific.

The synthesis on the support first provides a highly parallel and inexpensive peptide array for multiple uses.

6.3.1 Analyte An example for an analyte is a nucleic acid (DNA, RNA, in special cases PNA). These nucleic acids can be obtained from the whole genome, fragments thereof, chromosomes, plasmids or synthetic sources (eg cDNA). In one embodiment, the sample material is from the human genome.

Examples for further analytes are all forms of appearance, such as hormones, growth factors, enzymes, tumor antigens, serum factors, proteins, polypeptides and peptides of antibodies, hydrocarbons, such as various foods or crops Sugars, functional sugars, polymers and other organic molecules, such as drugs of abuse ', drugs, metabolites, amino acids, transmitters, chemical pesticides, pesticides, paints, various poisons, and the like.

6.3.2 Variant for Binding to an Immobilized Interaction Partner (Receptor) The binding of an analyte to a receptor is complementary in nucleic acid to nucleic acids, eg macromolecules, eg cDNA, synthetic. It can be performed by hybridization of oligonucleotide, PNA, and RNA. Peptides as receptors, such as synthetic or natural peptides, can bind to the analyte by protein-protein or protein-nucleic acid interactions.

6.3.3 Variants for Signal Generation Two principles for signal generation are advantageously used. That is: direct detection of the labeled analyte before or during the reaction (preferably a method of nucleic acid analysis by hybridization) and indirect detection of the analyte or target sequence by competition with a labeled standard. The first variant is well established for some uses, for example for the diagnosis of serum components, but is rather unsuitable and is also possible on supports using peptide arrays. The second variant is therefore advantageous in terms of use, and it also allows the user to prepare the sample in principle.

Direct detection involves dyes for absorbance measurement, labeling of the analyte with a fluorescent dye, labeling of the analyte with a reporter enzyme, and subsequent reactions (eg, chemiluminescence or bioluminescence).
Secondary detection of the bound analyte by selective labeling, eg, an inserted (fluorescent) dye, a double-stranded protein or a double-stranded antibody or a second component of the bound analyte. For example, in the case of a PNA-DNA hybrid, it can be carried out using a DNA-specific antibody. As the labeled standard, an enzyme-linked standard (for example, chemiluminescence and bioluminescence by alkaline phosphatase, peroxidase, etc.) or a (fluorescent) dye-conjugated standard can be used. Protein standards can be used as fusion proteins with reporter enzymes (see above) or autofluorescent proteins (eg, GFP), eg, for recombinant antibodies, protein-hormones, growth factors, and the like.

6.4 Preparation of sample material There are also various variants for the preparation of sample material. The method of preparation is not critical for detection of the body. This is because the DNA fragments always dissolved in the liquid at the contact point are prepared in an amount sufficient for the intended investigation.

6.4.1 External Sample Preparation Sample preparation can be performed manually in the laboratory on a separate analytical system or in a preparation device integrated into the same system. The sample ready for detection is introduced into the support by manual or automatic pipetting or by a comparable method.

6.4.2 Sample Preparation in the Same Support “All-in-One” Just as when using multiple uses to reduce production time in support synthesis, the same, or shorter, time is required for receptor synthesis. This is achieved, for example, when DNA amplification of a sample by PCR is inevitable. This makes the integration of PCR into a synthetic system or even a support reasonable for many uses.

In addition to time-intensive PCR, prior cell lysis can be integrated as well as DNA isolation, for example by well-automated methods such as ultrasound or high pressure.

6.5 Detection device The selection of the light signal for the detection reaction on the support array should be carried out in a detection device, wherein the excitation light source (fluorescence, emission or absorption for optical detection) ) Are placed directly opposite the CCD chip for measuring the light signal.
The support array is between the light source and the detection chip (sandwich type). An irradiation matrix is considered as the excitation light source. The spatial arrangement of the device can be performed as required (eg, using gravity for the flow process in the chip). The light path and the required light intensity are also reduced by the compact form as possible. The use of expensive position-intensive, highly light-absorbing optics should be abandoned not only on the excitation surface but also on the detection surface.

6.5.1 Temperature at Hybridization Temperature at hybridization (currently typically in the most recent developments at 60 ° C. already requires a small amount of salt to achieve hybridization at 25 ° C.) It can be implemented by itself by means of a corresponding temperature element in the device or an excitation light source or excitation light. Temperature elements on the support are likewise possible.

6.5.2 Excitation Light Source Excitation light may be selected depending on the label of the analyte (detection method via absorption or fluorescence).
Very parallel light from a lamp (white light), very parallel light from a discharge tube, very parallel monochromatic light, monochromatic laser beam, flat illumination by laser line expansion, monochromatic laser line or programmable light source matrix I do.

If appropriate, a corresponding optical grid or corresponding optics may be present between the excitation light source and the support array.

6.5.3 CCD Camera Detection The detection device preferably consists of only one CCD chip. They currently have about 2000 × 3000 pixels on a 25 × 37 mm surface (Cannon). When arranging about 500 parallel passages with a diameter of about 20 μm on such a surface of 25 × 37 mm (single-spaced double-pixel column), only one double-spaced pixel is used under the passage. In each case, 750 measurement points (areas) are obtained for each passage. At the same time, there are 375000 reaction areas on a single support, where each reaction area covers 4 color pixels or 12 black and white pixels and has a surface of 20 × 20 μm. The light signal must be generated as densely as possible on the optical CCD, so that the erroneous assignment of the light signal and the measuring point can be interrupted by its specific base sequence and the overlap of adjacent light signals. Otherwise, a serial detection of the overlapping area is performed or a fiber optic element is used.

Provided (current CCD chip technology) a corresponding number of measurements (0-4096)
4x500x750 = 1.5 Mio color signal or 4.5 Mio intensity value) form the basis that allows extensive statistics in the analysis of the detected light signal. The processing of the resulting data volume is possible without problems by price destruction at the same time with the development of the performance of modern computer systems.

The detection of the detection reaction can also be qualitatively and quantitatively described, in particular when the capture molecule (located in the array) finds an additional partner (eg using a fluorescently labeled label).
And find out how many hybridization partners the capture molecule class has.

If appropriate, a corresponding optical grid or a corresponding optical system may comprise a support array and C
It may be between the CD camera chips.

If detection by a CCD camera or CCD chip does not produce a sufficient signal, detection in the analysis system can also be performed by another sensitive sensor.

In the context of the present invention, we are interested in the use of inspection devices as described in German Patent Application No. DE 198 39 254.0. The inspection device has an electronically adjustable light source matrix, ie a light sensor matrix facing the light source matrix, ie a CCD image receiver.

In this regard, it is envisaged that the user will produce the support himself and use it directly. The user can easily load important data (DNA sequences) from CD-ROM or the Internet, and manufacture the individual DNA chips in the illuminated matrix CCD device, which are subsequently wetted with the sample and the signals are sorted out. .

In the above arrangement for photoactivity, for example, when measuring pixels at one interval,
In the meantime, the pixels in the projection inside the passage can be used for permanent process control. For this purpose, for example, bubbles may be individually and dynamically introduced between the two liquids in the passage. Also, since the colors of the support fluids for G, A, C and T are assumed,
The presence of the correct oligo can be checked, and the color change can signal the replacement. In subsequent detection, site-specific photoexcitation and, if necessary, color-specific photoexcitation can again be performed. This opens up entirely new possibilities for detection methods that currently do not exist.

With the aid of an inspection device (irradiation matrix-CCD device), the flow process in the passages in the support can be observed both during production (ie oligo synthesis) and during analysis. For this purpose, for example, the cleaning bubbles can use a coloring of two liquids in a plurality of passages or of individual liquids.

For the photo-induced separation of the protecting groups during the synthesis of the DNA oligos on or in the support, irradiation matrices generating and transmitting the required wavelengths, for example from 360 to 370 nm, are useful.

The detection of the detection reaction in the support can likewise be carried out in a test device. If detection by means of fluorescent labels is realized, this requires that the background illumination be switched if necessary (automatically possible), with optical filters and / or glass fiber elements (“Taper”) Can be used. In some cases, novel detection methods can also be used here, which are possible firstly by extremely flexible individual irradiation and detection of individual reaction areas.

A temperature of about 55-65 ° C. is required for standard hybridization of the DNA, RNA and PNA strands to each other. Most simply, this temperature is generated by the irradiation energy of the irradiation matrix (waste heat and wavelength). This allows further miniaturization of the arrangement.

[0132] 8. Illustrative Embodiments Synthesis of DNA molecules in multiple pathways can be used with a suitable protecting group, such as dimethoxymethyl (DMT), using a standard synthon, such as a Phosphoramidit component. Starting from a linker coupled to a solid phase, the corresponding liquid DNA synthesis can be performed.

This level may be combined with the light-dependent control of DNA synthesis for an advantageous embodiment according to the invention. Protecting groups which allow light-dependent deprotection in that regard are known, so that mainly those protecting the 5 'carbon atom of the synthon are deprotected by light of the appropriate wavelength. As described above, it is possible to synthesize a nucleic acid having a length of 18 nucleotides or more in a capillary tube.

As is possible by the separation of the synthesized DNA oligomers and by the use of suitable linkers, the reaction products are, for example, high performance liquid chromatography (HP
LC). In this case, the capillary DN depends on the proportion of full-length product.
The efficiency of A synthesis can be shown.

For light-dependent DNA synthesis, the reaction area on the support is regiospecifically and / or time-specifically using a suitable light source, for example a mercury vapor lamp, laser light (eg having 373 nm). (Nitrogen laser) or UV-LED. Similarly, light sources with sufficiently high energy radiation are also suitable.

FIG. 1 shows a support according to the invention in a very simplified illustration.

FIG. 2 shows an example for a passage arrangement in a support according to the invention.

FIG. 3 shows a schematic diagram of a support in an inspection device consisting of a programmable light source matrix and a CCD matrix.

FIG. 4 shows a schematic diagram of an apparatus according to the invention for a light-based integrated synthesis and analysis method.

FIG. 5 shows the arrangement from FIG. 4 for the individualization of the liquid in the reaction zone.

FIG. 1 shows the transparent support in a very simplified diagram. Paths running parallel to each other, for example 500 paths with a length of 37 nm are identified. T, G
, A, C indicate the storage for the individual substances used (bases). 3
Indicates the gas inlet. Reference numeral 5 denotes a valve. Numeral 7 indicates sample loading and 9 indicates an inlet for other synthetic chemicals as well as cleaning / washing solutions.

FIG. 2 shows a further example for selective passage arrangement.

FIG. 3 shows a programmable light source matrix such as an LCD matrix and a CC.
2 shows a support according to FIG. 1 in an inspection device comprising a D detection matrix.

FIG. 4 shows a device according to the invention with a replaceable support 40, the basic configuration being such that the support is replaceable or exhausted in each cycle. If you do, don't rely on it. In the final case, the cleaning and subsequent reuse of its own aisle is carried out. Reference numeral 30 denotes a programmable light source matrix. Since its programmability is built into a system component 20 consisting of a computer or a computer, only one freely attachable light source matrix is required as component 30. The light source matrix 30 irradiates light of a specified wavelength and intensity to an arbitrary position in at least a two-dimensional matrix. The light source matrix 30 serves for very parallel irradiation of the reaction area on the support 40. The support 40 is individually illuminated in all reaction regions with a light pattern composed of energy waves that is computer-controlled by the light source matrix 30. Via the liquid connection system 64, the liquid prepared from the liquid module 60 is transported to the support 40 and re-transmitted in a suitable manner into the support microstructure, not shown, to the reaction zone. This makes the support 40 an optical fluid microprocessor. It can be replaced for each use, or it can be cleaned for each use and replaced for practical purposes only when exhausted.

Incident light can be used, for example, for absorbance measurements where activation of a photoreaction or excitation of fluorescence is used.

Light incident from the support 40 or into the optofluidic microprocessor may be, for example, light passing through the support of the light source matrix 30 in transmitted light. However, this can be a light signal, which gives rise to, for example, fluorescence or luminescence at individual reaction areas of the support 40.

The detection matrix 50, for example, consisting of a CCD chip with or without optics, has a support 40 facing and between the light source matrix 30, so that a triple matrix arrangement is achieved with the light matrix. , A support and a detection matrix.

The liquid module 60 serves for the supply of the reaction support 40, for example, for the use substances, protective gases, chemical substances, for example solvents and the like, and for sample material. Liquid module 60
Consists of a tank 61, which is emptied in a suitable manner by a pump 62 and a valve 63. The tanks can be replaced individually or collectively or refilled. Permanently required liquids, such as protective gas, can be supplied continuously (without tanks in the system) by conduits. The liquid waste of the various methods can be received in an integrated tank in the support 40, or in a waste system 65, or collectively outside the individual systems.

Similarly, the boundaries 10 of the system of the device are represented. The devices can also be used in individual instruments or in centralized or decentralized clusters. These clusters are always connected to each other by information technology. The one-point system can supply energy, liquids such as materials used, reactive chemicals, protective gases and sample materials, and essential supports, either manually or by automated components together.

A system component 20, in the form of a computer or computer, is responsible for controlling or regulating the system. There, the light source matrix 30 and the liquid components 60 are controlled for the individual reaction zones based on the calculation of the probe array or the receptor array. Furthermore, the data of the detection matrix 50 is considered and evaluated.

Each device may therefore be in communication with another device or another system or another computer or database of devices according to the present invention across its system boundary 10. This may take place, for example, via wires, bus systems or the Internet. In this case, the communication can take place centrally via the main computer or as a cluster of equivalent systems. Similarly, the data interface is provided in the system environment in advance.

FIG. 5 shows the arrangement from FIG. 4 for the individualization of the liquid in the reaction zone. Reference numeral 41 also indicates a support. It is individually wetted under computer control by a liquid deprotection module 32. Via the liquid connection system 64, the liquid prepared from the liquid module 60 is transported to the support and is retransmitted to the reaction zone in a manner appropriate to the support microstructure not shown. This makes the support 41 an optical fluid microprocessor. It may be replaced for each use, or it may be cleaned for each use and replaced when worn for practical purposes.

In the support, light for excitation such as a fluorescence reaction may be supplied from above and / or side.

Light incident from the support or into the optofluidic microprocessor can be generated, for example, by illuminating the reaction area.

The liquid deprotection module 32 has at least one reaction region on each support 41.
The liquid can be brought into contact individually with the three wet components 33 (for example, nozzles, capillaries, etc.). This can, for example, locally activate chemical and biochemical reactions.

The liquid module 31 serves for the supply of the deprotection module 32 with a liquid having the use substance or the chemical substance. The liquid module 31 has the same configuration as the module 60, and includes a tank, a conduit, a valve, and the like as necessary.

The detection matrix 50, for example composed of a CCD chip with or without optics, has a support 41 facing and in between the protection module 32 by liquid, and thus a triple matrix arrangement. Arranged to occur.

The liquid module 60 serves for supplying the support 41 with, for example, substances to be used, protective gases, chemicals, for example solvents, etc. as well as sample materials. The liquid module 60 comprises a tank 61, which is emptied in a suitable manner by a pump 62 and a valve 63. The tanks can be replaced individually or collectively, or can be refilled. Permanently required liquids, such as protective gas, can be supplied continuously by a conduit (no tank in the system). The liquid waste of the various processes can be received in tanks incorporated in the support 41 or in the waste system 65 or collectively outside the individual systems.

The system boundaries 10 of the device already described are also shown, and the system components 20 are shown in the form of a computer or a computer that takes over the control or regulation of the system. This allows the liquid modules 31 and 60 and the liquid deprotection module 3 to be based on the calculation of the probe arrangement for the individual reaction areas.
2 is controlled. Furthermore, the data of the detection matrix 50 is considered and evaluated.

Each device may thus be in communication with another device or a system of devices according to the invention or another computer or data bank across its system boundary 10. This may take place, for example, via wires, bus systems or the Internet. In this case, the communication takes place centrally via the main computer or as a cluster of equivalent systems. Similarly, data interface 2
1 is provided in the system environment in advance.

[Brief description of the drawings]

FIG. 1 shows a support according to the invention in a very simplified illustration.

FIG. 2 shows an example for a passage arrangement in a support according to the invention.

FIG. 3 shows a schematic view of a support in an inspection device consisting of a programmable light source matrix and a CCD matrix.

FIG. 4 shows a schematic diagram of an apparatus according to the invention for a light-based integrated synthesis and analysis method.

FIG. 5 shows the arrangement from FIG. 4 for the individualization of the liquid in the reaction zone.

[Explanation of symbols]

1 passage, 3 gas inlet, 5 valve, 7 sample input, 9 inlet of other synthetic chemicals and cleaning / washing liquid, 10 system boundary, 20 system components, 21 data interface, 30 light source matrix, 31
Liquid module, 32 Deprotection module, 33 Wet component, 40, 41
Support, 50 detection matrix, 60 liquid modules, 61 tanks, 62 pumps, 63 valves, 64 liquid connection systems, 65 waste systems, T, G, A, C repositories.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] December 4, 2000 (2000.12.4)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme court ゛ (Reference) G01N 37/00 102 C12N 15/00 AF (31) Priority claim number 198 39 255.9 (32) Priority Date August 28, 1998 (August 28, 1998) (33) Priority claim country Germany (DE) (31) Priority claim number 199 07 080.6 (32) Priority date February 19, 1999 (February 19, 1999) (33) Priority claim country Germany (DE) (31) Priority claim number 199 24 327.1 (32) Priority date May 27, 1999 (May 27, 1999) ( 33) Priority claim country Germany (DE) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), AU, CA, JP, US (72) Inventor Manfred Müller Germany Munich Reuters Strasse 76 / Bee (72) Inventor Fritz Steller Germany Weinheim Gösselweg 15 (72) Inventor Hans Lintner Germany Germany Schuttgart Wielleichweg 27 F term (reference) 4B024 AA11 AA20 HA11 HA19 4B029 AA23 BB20 CC03 FA10 4B063 QA01 QA13 QQ42 QQ52 QR31 QR82 QS34 QX02

Claims (38)

[Claims] 1. A step of: (a) providing a support having at least one passage; and (b) directing a liquid having components for the synthesis of a polymer receptor through one or more passages of the support. (C) immobilizing the receptor component at each predefined location in one or more passages in a position-specific and / or time-specific manner; (d) the desired receptor is defined at each predefined location. Process until it is synthesized at the position (
A method for producing a support for measuring an analyte, comprising the steps of repeating b) and (c). 2. The method according to claim 1, wherein a support is produced having defined surface areas each having the same receptor type. 3. The method according to claim 1, wherein a plurality of passages are arranged on at least one support surface. 4. The method as claimed in claim 1, wherein the support has a plurality of passages, which are preferably arranged parallel to one another. 5. The method according to claim 1, wherein the receptor is selected from nucleic acids and nucleic acid analogues. 6. The method according to claim 5, wherein the receptor component is selected from nucleotides, oligonucleotides, nucleotide analogs and oligonucleotide analogs. 7. The method according to claim 1, wherein the receptor is selected from a polypeptide. 8. The method according to claim 7, wherein the receptor component is selected from amino acids and peptides. 9. The method according to claim 1, wherein the position-specific and / or time-specific immobilization of the receptor component is carried out by irradiation. 10. The method according to claim 9, wherein the illumination is performed by a programmable light source matrix. 11. The method according to claim 1, wherein the position-specific and / or time-specific immobilization of the receptor component is carried out by wetting the activated position with an activated liquid under controllable selection of the activated position. The method according to any one of the preceding claims. 12. The method according to claim 1, wherein the pattern of the polymer receptor is defined by a computer program. 13. The method according to claim 1, wherein the support is used for measuring an analyte in a sample. 14. The following steps: (a) preparing a support; (b) introducing a liquid containing a receptor or a component onto the support to synthesize a polymer receptor; (c) on a support Immobilizing the receptor or receptor component at each predefined location in a position-specific and / or time-specific manner, (d) optionally allowing the desired receptor to be located at each predefined location on the support. Repeating steps (b) and (c) until synthesized; (e) contacting the support with a sample containing the analyte; (f) by binding to a receptor immobilized on the support. A method for integrated synthesis and analytical measurement on a support, comprising the step of measuring an analyte. 15. The method according to claim 14, wherein the synthesis and the analytical measurement are performed in an integrated device, wherein the synthesis and / or the analytical measurement process is monitored and adjusted at any number of positions on the support. the method of. 16. A programmable light source matrix, a detection matrix, a support disposed between the light source matrix and the detection matrix, and a means for supplying liquid into the support and for discharging liquid from the support. 16. The method according to claim 15, wherein an integrated device is used, including means. 17. The method according to claim 14, wherein the analyte is removed from the support again after the measurement. 18. The method according to claim 14, wherein several synthesis / analysis measurement cycles are performed, wherein the receptors for the following cycles are synthesized on the basis of information from the preceding cycle. The method described in the section. 19. The method of claim 18, wherein the method is performed for a subsequent cycle of elongation of the receptor from a previous cycle. 20. The method of claim 18, wherein a new support having a receptor modified for a preceding cycle relative to a preceding cycle is synthesized. 21. The method of claim 20, wherein altering the receptor eliminates alterations in the sequence and / or negative receptors of the preceding cycle. 22. The method according to claim 14, wherein a flat support is used. 23. The method according to claim 14, wherein a support having a plurality of passages is used.
The method according to any one of the preceding claims. 24. The method according to claim 14, wherein several supports are used for the synthesis / analytical measurement cycle. 25. Synthesizing and analyzing several supports in various detection devices which are connected to one another in information technology but may be spatially separated from one another.
The method according to claim 24. 26. A support having a plurality of passages, in particular a capillary passage, is used for the determination of an analyte in the method according to claim 13, wherein
Use wherein a number of different receptors are immobilized in a plurality of passages. 27. The use according to claim 26, wherein the support is optically transparent at least in the region of the reaction zone. 28. A reagent comprising a support according to any one of claims 26 or 27 and a component for the synthesis of a polymer receptor on a support in the method according to any one of claims 13 to 25. Use of the kit. 29. A programmable light source matrix, a detection matrix, a support disposed between the light source matrix and the detection matrix, and means for supplying a liquid into the support and for discharging a liquid from the support. 26. Use of a device for integrated synthesis and analytical measurement on a support comprising means in a method according to any one of claims 15 to 25. 30. The use according to claim 29, wherein the apparatus further comprises means for deprotection of the reactants on the support. 31. Use according to claim 29 or claim 30, wherein the device further comprises electronic control and adjustment means. 32. The method according to any one of claims 1 to 25, the support according to claim 26 or 27, the reagent kit according to claim 28, or the reagent kit according to any one of claims 29 to 31. The use of the device of claim 1 for the determination of an analyte in a sample. 33. Use according to claim 32 for sequencing nucleic acids. 34. The use according to claim 33, for the novel sequencing and / or resequencing of complex genetic material, for example individual genomic or synthetic nucleic acids. 35. The use according to claim 32, for the management of an individual patient, for example to obtain diagnostic information for the individual action of a drug. 36. Use according to claim 32 for the analysis of the action of pharmacological substances. 37. The method according to claim 32, for preparing and analyzing a substance library.
Use of the description. 38. The use of claim 32 for comparing analytes in a population.